Biology subjects

Paulo, J. A.

Publications and source records attributed to Paulo, J. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Harmine Selectively Drives Human Beta Cell Differentiation and Function Via Protein Kinase A Pathways

Harmine and other small molecule inhibitors of the kinase DYRK1A induce human beta cells to replicate and regenerate in vitro and in vivo, and are effective at reversing diabetes in animal models. In addition to its beta cell proliferative and regenerative effects, harmine also induces expression of transcription factors and other genes involved in beta cell differentiation and function, exemplified by PDX1, MAFA, NKX6.1, GLP1R, PCSK1 and many others. Harmine also rapidly enhances glucose-stimulated insulin secretion in vitro and in vivo, reversing diabetes within days in diabetic mice transplanted with a marginal mass of human islets. We had assumed that this pro-differentiation or pro-function effect was a common feature of all DYRK1A inhibitors, and was mediated by DYRK1A inhibition. Thus, DYRK1A is a primary target (we refer to it as Target 1) for harmine. Here, to our surprise, we report that the pro-differentiation effect is not a generalized action for all small molecule DYRK1A inhibitors and does not result from DYRK1A interference or genetic silencing. Instead, the prodifferentiation effect is restricted to a small select subset of DYRK1A inhibitors (harmine, 2-2c and 5-IT). Remarkably, the pro-differentiation effect results from the unique ability of these drugs to activate protein kinase A (PKA), a dual mechanism that distinguishes this class from other DYRK1A inhibitors. The beneficial effects of harmine on PKA appear to be indirect, driven by an as yet unidentified, Target 2 in the PKA pathway. These findings make it clear that all DYRK1A inhibitors are not interchangeable, and that those that drive both proliferation and differentiation/function will likely be preferable in human clinical therapeutic settings. They also provide a novel target or roadmap for enhancing human beta cell differentiation in Type 1 and Type 2 diabetes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/692818v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@bd015corg.highwire.dtl.DTLVardef@a46f9borg.highwire.dtl.DTLVardef@19b4f65org.highwire.dtl.DTLVardef@179aa28_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Shifted assembly and function of mSWI/SNF family subcomplexes underlie targetable dependencies in dedifferentiated endometrial carcinomas

The mammalian SWI/SNF (mSWI/SNF) family of chromatin remodelers govern cell type-specific chromatin accessibility and gene expression and assemble as three distinct complexes: canonical BAF (cBAF), Polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF). ARID1A and ARID1B are paralog subunits that specifically nucleate the assembly of cBAF complexes and are frequently co-mutated in highly aggressive dedifferentiated/undifferentiated endometrial carcinomas (DDEC/UECs). Here, in cellular models and primary human tumors, we find that ARID1A/B deficiency-mediated cBAF loss results in increased ncBAF and PBAF biochemical abundance and chromatin-level functions to maintain the DDEC oncogenic state. Further, treatment with clinical-grade SMARCA4/2 ATPase inhibitors markedly attenuates DDEC cell proliferation and tumor growth in vivo and synergizes with carboplatin-based chemotherapy to extend survival. These findings reveal the oncogenic contributions of shifted mSWI/SNF family complex stoichiometry and resulting gene regulatory dysregulation and suggest therapeutic utility of mSWI/SNF small molecule inhibitors in DDEC/UEC and other cBAF-disrupted cancer types.

molecular biology↗

bioRxiv2025

Proteome Landscapes Decode Organelle Vulnerabilities in cortical and dopaminergic-like induced neurons Across Lysosomal Storage Disorders

Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy, and by recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) comprise a group of diseases affecting diverse lysosomal functions. To facilitate molecular phenotyping across diverse LSD gene classes, we are developing a library of human embryonic stem cells engineered to lack individual LSD genes as a resource for the field. Here, we report our initial stem cell toolkit lacking one of 23 LSD genes, including the majority of genes associated with sphingolipidoses and neuronal ceroid lipofuscinoses, and its use in the generation of a proteomic resource for induced cortical-like and midbrain dopaminergic-like neurons In-depth abundance and correlation profiling across organelles and sub-organelle components revealed potential vulnerabilities that reflect distinct patterns of proteome alterations across both genotypes and neuronal cell types. We characterize alterations in the mitochondrial proteome associated with GBA1 and ASAH1 deficiency and identify synaptic and mitochondrial defects in ASAH1-/- induced neurons that correlate with defects in neuronal firing rates. Moreover, we developed an informatic pipeline for proteome-wide identification of individual protein interactions and protein complexes that may be disrupted as a result of LSD gene deficiency. Finally, we visualized structural alterations of ASAH1-deficient endolysosomes in situ using cryo-electron tomography, revealing swollen organelles that were largely devoid of dense internal membranes characteristic of wild-type cells, but containing numerous intralumenal vesicle compartments. This toolkit and associated proteomic landscapes provide a resource for defining molecular signatures associated with LSD gene dysfunction and organelle vulnerability.

cell biology↗

bioRxiv2025

Promoter hijacking by primate LINC00473 disrupts an ancestral CREB-PDE10A feedback loop

Thousands of human long noncoding RNAs (lncRNAs) evolved in primates, although much remains unknown about how these lncRNAs shaped human gene regulatory networks. The primate-specific lncRNA gene LINC00473 has a CREB-inducible promoter that is conserved beyond primates and the gene is located upstream of PDE10A, which encodes an inhibitor of CREB. To gain insight into the regulatory consequences of LINC00473 acquisition, we tested the cellular function of the conserved mouse promoter. We found that the homologous mouse promoter is induced by CREB and regulates the downstream Pde10a gene in C2C12 myoblast-like cells and neurons. Activation of this promoter by CRISPRa increased Pde10a transcript and protein levels, and proteomics revealed that elevated Pde10a promoted C2C12 differentiation at the expense of proliferation. Activation of Pde10a by CRISPRa also impaired CREB-dependent gene expression, suggesting that the mouse homolog of the LINC00473 promoter drives a CREB-Pde10a feedback loop. In contrast to the mouse homolog, human LINC00473 promoter activation by CRISPRa increased LINC00473 expression with either no change in PDE10A or delayed induction compared to mice. Our findings suggest that the newly evolved LINC00473 gene hijacked an ancestral CREB-inducible Pde10a promoter, thereby disrupting a negative feedback loop that may otherwise constrain CREB-dependent gene expression. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/680088v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1862846org.highwire.dtl.DTLVardef@be3ca8org.highwire.dtl.DTLVardef@1806b36org.highwire.dtl.DTLVardef@1fedc56_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Next-Generation Multiplexed Targeted Proteomics Quantifies Post-Translational Modifications, Compound-Protein Interactions, and Disease Biomarkers with High Throughput

The GoDig platform enables sensitive, multiplexed targeted pathway proteomics without manual scheduling or synthetic standards. Here we present GoDig 2.0, which increases sample multiplexing to 35-fold, improves time efficiency and reduces scan delays for higher success rates, and allows flexible spectral and elution library generation from different mass spectrometry data types. GoDig 2.0 measures 2.4x more targets than GoDig 1.0, quantifying >99% of 800 peptides in a single run. We compiled a library of 23,989 human phosphorylation sites from a phosphoproteomic dataset and used it to profile kinase signaling differences across cell lines. In human brain tissue, we established a hyperphosphorylated tau assay including pTau127, revealing potential biomarkers for Alzheimers disease. We also quantified diglycyl-lysine peptides to assess polyubiquitin branching. Finally, we built a library of 20,946 reactive cysteines and profiled covalent compound-protein interactions spanning diverse pathways. GoDig 2.0 enables high-throughput analyses of site-specific protein modifications across many biological contexts.

biochemistry↗

bioRxiv2025

HuD controls widespread RNA stability to drive neuronal activity-dependent responses

Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism in vitro and in vivo. Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for [~]15% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Catalytic pocket of Clr4 (Suv39h) methyltransfer-ase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

Histone H3 lysine 9 (H3K9) methylation must be regulated to prevent inappropriate heterochromatin formation. Regulation of the conserved fission yeast H3K9 methyltransferase Clr4 (Suv39h) involves an automethylation-induced conformational switch and interaction of its catalytic SET domain with mono-ubiquitinated histone H3 lysine 14 (H3K14ub), a modification catalyzed by the Cul4 subunit of the CLRC complex. Using reconstituted CLRC, we show that Clr4 catalytic pocket serves as a substrate receptor for Cul4-dependent H3K14 ubiquitination. H3K14ub activates Clr4 to catalyze cis methylation of H3K9 on the same histone tail, while Clr4 auto-methylation enables H3K14ub-bound Clr4 to methylate H3K9 on an unmodified H3 tail in trans. Crosslinking and structural modeling reveal interactions between Clr4 chromo and SET domains, and between the chromo-domain and H3K14ub, suggesting that the chromodomain reads H3K9me3 and H3K14ub to allosterically regulate Clr4 activity. H3K14 ubiquitination therefore regulates Clr4 by promoting its recruitment and by positioning H3K9 in the active site.

biochemistry↗

bioRxiv2025

A SWI/SNF-specific Ig-like domain, SWIFT, is a transcription factor binding platform

Mammalian SWI/SNF (BAF) chromatin remodeling complexes modulate DNA accessibility and gene expression, however, their genomic targeting mechanisms remain incompletely understood. Here, we identify SWIFT (SWI/SNF Ig-Fold for Transcription Factor Interactions), a conserved, broad transcription factor (TF) binding platform on the SMARCD subunits. SWIFT is necessary and sufficient for direct engagement with the transactivation domain of PU.1, a single mutation in which disrupts PU.1-mSWI/SNF binding, impairs complex targeting, and attenuates oncogenic transcription and proliferation in PU.1-dependent cancer cells. Dominant expression of the SWIFT domain in isolation sequesters TFs from mSWI/SNF and poisons TF-addicted cancer cells. Finally, TFs across diverse families interact with SMARCD paralog-specific SWIFT domains. These results define a major mechanism of cell type- and disease-specific mSWI/SNF chromatin targeting and inform approaches toward therapeutic modulation.

molecular biology↗

bioRxiv2025

Integral Synthesis and Clearance Analysis via DIA (ISDia) Reveals Coordinated Protein Dynamics Regulation during Endoplasmic Reticulum Stress

Endoplasmic Reticulum (ER) stress disrupts protein homeostasis, driving cellular responses critical for survival, development and disease. However, systematic proteome-wide analyses of protein synthesis and clearance during ER stress remain limited. In this study, we developed Integral Synthesis and clearance analysis via DIA (ISDia), a robust mass spectrometry (MS)-based workflow that integrates multiple pulsed-SILAC labeling timepoints under one condition into a single sample and utilize data-independent acquisition (DIA) to quantify heavy and light peptide changes across conditions. It enables proteome-wide tracking of protein synthesis and clearance with high coverage by directly comparing light and heavy peptides in pSILAC-DIA experiments across multiple conditions. Using ISDia, we uncover diverse regulatory mechanisms by which protein synthesis and clearance are modulated to regulate protein abundances during ER stress, revealing PERK dependent and independent regulatory mechanisms across subcellular compartments, complexes and isoforms. These findings highlight the potential of ISDia as a powerful and widely applicable workflow for revealing protein abundance regulatory mechanisms.

systems biology↗

bioRxiv2025

Baseline expression of c-Myc defines the tissue specificity of oncogenic K-Ras

KRAS is among the most frequently mutated oncogenes in cancer. Yet, mutations in KRAS are common only in tumors originating from a subset of tissues. It is critical to understand the molecular mechanisms underlying this oncogene tissue specificity. Utilizing genetically engineered mouse models carrying a conditional oncogenic allele of Kras, we expressed activated K-Ras in adult tissues to investigate its specificity. We discovered that the ability of K-RasG12D to influence the fitness of cells in a given tissue is not determined by its canonical signaling through MAPK. Instead, low baseline expression of c-Myc renders tissues non-permissive to oncogenic K-Ras, a context that can be reversed in the liver by ectopically expressing c-Myc. This functions independently of the proliferative index of the tissue or the induction of cell cycle arrest or apoptosis. Our findings reveal the importance of the basal state of the tissue-inherent signaling network for determining oncogene specificity.

cancer biology↗

bioRxiv2025

Cell-surface proteomic profiling identifies CD72 as a regulator of microglial tiling

Microglial tiling--the phenomenon of consistent cell-to-cell distances and non-overlapping processes--is regarded as a qualitative indicator of homeostasis, but mechanisms of microglial tiling are unknown. We used cell-surface proximity labeling and mass spectrometry to profile the microglial cell-surface proteome in an in vitro model of homeostatic glial physiology and used single-cell RNA sequencing and public databases to identify candidate cell-surface proteins that might modulate tiling. We designed an image-based functional assay which measures six morphological/spatial readouts to screen these proteins for modulation of tiling. CD72, a coreceptor to the B cell receptor that is expressed by microglia, disrupted tiling; we validated its effects in vitro and in situ in organotypic hippocampal brain slices. Phosphoproteomic studies revealed that CD72 modulates pathways associated with cell adhesion, repulsive receptors, microglial activation, and cytoskeletal organization. These results lay the groundwork for further investigation of the functional roles of tiling in homeostasis and disease.

neuroscience↗

bioRxiv2025

Creatine synthesis is a tumor suppressor pathway hypostatic to one-carbon metabolism

Methylene tetrahydrofolate reductase 2 (MTHFD2), the rate-limiting enzyme of mitochondrial one-carbon metabolism, is one of the most highly expressed metabolic enzymes across diverse cancers and lymphoproliferative disorders. However, its exact roles in oncogenic metabolism remain poorly defined. We show that MTHFD2 is a key regulator of mitochondrial energetics in Epstein-Barr virus-transformed B lymphoblastoid cell lines (LCLs), an in vitro model of post-transplant lymphoproliferative disorder (PTLD). We also delineate a role for MTHFD2 in fueling de novo creatine synthesis; MTHFD2 mediates the production of glycine, a necessary substrate for creatine synthesis, through serine catabolism. Aminomethyltransferase (AMT) suppression short-circuits the glycine cleavage system (GCS) to augment LCL mitochondrial glycine levels. Creatine synthesis is hypostatic to mitochondrial one-carbon metabolism; inhibition of creatine synthesis improves LCL fitness only when MTHFD2 is lost. Our findings emplace MTHFD2 at the nexus of amino acid and energy metabolism pathways in LCLs, with potential clinical ramifications for PTLD. Highlights* Complete activation of creatine synthesis in an in vitro cellular model of PTLD * Creatine synthesis is a major sink for mitochondrial 1C-derived glycine * Reverse GCS activity due to AMT deficiency in lymphoblastoid cells * Epistasis between mitochondrial 1C metabolism and creatine synthesis eTOC BlurbLeung et al. demonstrate that MTHFD2 is crucial for creatine synthesis in lymphoproliferative disorders. MTHFD2 supports forward 1C flux through SHMT and drives reverse GCS activity to augment mitochondrial glycine, a substrate for creatine synthesis. Tumor-suppressive effects of creatine synthesis are unmasked with MTHFD2 loss, exhibiting metabolic epistasis.

molecular biology↗

bioRxiv2025

Deazaguanylation is a nucleobase-protein conjugation required for type IV CBASS immunity

7-deazapurines are nucleobase analogs essential for nucleic acid modifications in nearly all cellular life. Here, we discover a role for 7-deazapurines in protein modification within type IV CBASS anti-phage defense and define functions for CBASS ancillary proteins Cap9 and Cap10 in nucleobase-protein conjugation. A structure of Cap10 reveals a tRNA transglycosylase-family enzyme remodeled to bind the modified N-terminus of a partner cGAS/DncV-like nucleotidyltransferase linked to a 7-amido-7-deazaguanine (NDG) nucleobase. The structure of Cap9 explains how this QueC-like enzyme co-opts a 7-deazapurine biosynthetic reaction mechanism for NDG conjugation. We demonstrate that Cap9, Cap10, and NDG conjugation are essential for host defense against phage infection. Our results define a previously unknown 7-deazapurine protein modification and explain how nucleobase biosynthetic machinery has been repurposed for antiviral immunity.

microbiology↗

bioRxiv2025

Muscle-specific increased expression of JAG1 improves skeletal muscle phenotype in dystrophin-deficient mice.

Therapeutic strategies for Duchenne Muscular Dystrophy (DMD) will likely require complementary approaches. One possibility is to explore genetic modifiers that improve muscle regeneration and function. The beneficial effects of the overexpression of Jagged-1 were described in escaper golden retriever muscular dystrophy (GRMD) dogs that had a near-normal life and validated in dystrophin-deficient zebrafish (1). To clarify the underlying biology of JAG1 overexpression in dystrophic muscles, we generated a transgenic mouse (mdx5cv-JAG1) model that lacks dystrophin and overexpresses human JAG1 in striated muscles. Skeletal muscles from mdx5cv-JAG1 and mdx5cv mice were studied at one, four, and twelve-month time points. JAG1 expression in mdx5cv-JAG1 increased by three to five times compared to mdx5cv. Consequently, mdx5cv-JAG1 muscles were significantly bigger and stronger than dystrophic controls, along with an increased number of myofibers. Proteomics data show increased dysferlin in mdx5cv-JAG1 muscles and an association of Nsd1 with the phenotype. Our data supports the positive effect of JAG1 overexpression in dystrophic muscles. Significance StatementDuchenne Muscular Dystrophy (DMD) patients present a progressive decline in motor function. DMD is caused by mutations in the DMD gene that lead to the absence of dystrophin - an essential component of muscle cells. However, dystrophin-deficient dogs overexpressing JAG1 had a normal lifespan with remarkable motor function. In this study, we increased expression of human JAG1 in mouse skeletal muscles lacking dystrophin to explore mechanisms responsible for these benefits. Our observations show that overexpression of JAG1 counterbalances the lack of dystrophin by generating bigger and stronger muscles as the mouse ages. Moreover, our proteomics dataset suggests a role of dysferlin in the phenotype. Therefore, our study supports the exploration of JAG1 in pre-clinical models.

physiology↗

bioRxiv2025

An RNA Splicing System that Excises Transposons from Animal mRNAs

All genomes harbor mobile genetic parasites called transposable elements (TEs). Here we describe a system, which we term SOS splicing, that protects C. elegans and human genes from DNA transposon-mediated disruption by excising these TEs from host mRNAs. SOS splicing, which operates independently of the spliceosome, is a pattern recognition system triggered by base-pairing of inverted terminal repeat elements, which are a defining feature of the DNA transposons. We identify three factors required for SOS splicing in both C. elegans and human cells; AKAP17A, which binds TE-containing mRNAs; the RNA ligase RTCB; and CAAP1, which bridges RTCB and AKAP17A, allowing RTCB to ligate mRNA fragments generated by TE excision. We propose that SOS splicing is a novel, conserved, and RNA structure-directed mode of mRNA splicing and that one function of SOS splicing is to genetically buffer animals from the deleterious effects of TE-mediated gene perturbation.

genetics↗

bioRxiv2025

The role of MKI67 in the regulation of 60S pre-ribosome nucleolar export, transcripts, energy supply, and apoptosis

Ki-67, a protein encoded by the MKI67 gene, is present in proliferating human tissue cells. It is an important marker for diagnosing cancer. Despite numerous investigations, the function of Ki-67 remains unclear. In this study, we examined the role of Ki-67 and its associated proteins, GNL2 and MDN1, in the HEK293T neuronal lineage cell line using ChIP-seq, RNA-seq, confocal microscopy, and biochemical methods. Ki-67 bound to nearly all regions of the chromosomes through certain consensus nucleotide sequences and was primarily present at the nucleolar periphery, where it interacted with GNL2. These two proteins could recruit MDN1 to the periphery. Depleting Ki-67, GNL2, or MDN1 resulted in characteristic changes in nucleolar protein and chromatin localization. These phenotypes and protein-protein interaction data suggest that Ki-67 regulates the import of nuclear proteins and the export of pre-60S particles. Specifically, Ki-67 retained pre-60S particles within the nucleolus and, when recruiting MDN1, released them along with itself and chromatin. Depleting any one of these proteins decreased the levels of RNAs involved in ribosome biogenesis, with the strongest decrease occurring with MDN1 depletion. MDN1 depletion also decreased transcripts involved in mitochondrial respiration, resulting in strong culture acidification. Conversely, depleting Ki-67 increased the levels of over a thousand different transcripts. The greatest number and strongest increases were of transcripts necessary for neuronal development and activity. These included PAX5, SCG3, UNC13A, SLITRK1, CHGB, CHGA, POU4F2, KCNB1, NEK7, and SLC7A2. SLC7A2 encodes an amino acid transporter that activates mTORC1 with imported amino acids. However, the transcript of NEUROG2, a master regulator that activates downstream genes necessary for forming neuronal structures, did not increase. Taken together, these results suggest that Ki-67, GNL2, and MDN1 promote neuronal cell proliferation by regulating ribosome processing and energy production. Nevertheless, the disappearance of Ki-67 is essential for nervous system development, activity, and survival.

cell biology↗

bioRxiv2025

EndoMAPV1, a Structural Protein Complex Landscape of Human Endosomes

Early/sorting endosomes are dynamic organelles that play key roles in proteome control by triaging plasma membrane proteins for either recycling or degradation in the lysosome1,2,3. These events are coordinated by numerous transiently-associated regulatory complexes and integral membrane components that contribute to organelle identity during endosome maturation4. While a subset of the several hundred protein components and cargoes known to associate with endosomes have been studied at the biochemical and/or structural level, interaction partners and higher order molecular assemblies for many endosomal components remain unknown. Here, we combine cross-linking and native gel mass spectrometry5-8 of purified early endosomes with AlphaFold9,10 and computational analysis to create a systematic human endosomal structural interactome. We present dozens of structural models for endosomal protein pairs and higher order assemblies supported by experimental cross-links from their native subcellular context, suggesting structural mechanisms for previously reported regulatory processes. Using induced neurons, we validate two candidate complexes whose interactions are supported by crosslinks and structural predictions: TMEM230 as a subunit of ATP8/11 lipid flippases11 and TMEM9/9B as subunits of CLCN3/4/5 chloride-proton antiporters12. This resource and its accompanying structural network viewer provide an experimental framework for understanding organellar structural interactomes and large-scale validation of structural predictions.

cell biology↗

bioRxiv2024

CEBPA repression by MECOM blocks differentiation to drive aggressive leukemias

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, yet the mechanisms through which this occurs remain poorly understood. Increased expression of the stem cell transcription factor, MECOM, underlies one key driver mechanism in largely incurable AMLs. How MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem cell-like states by directly repressing pro-differentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kb downstream of the myeloid differentiation regulator CEBPA, is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo, suggesting a broadly applicable differentiation-based approach for improving therapy.

cancer biology↗